Visualization of the VBC complex in living cells by FLIM-FRET
| Project/Area Number |
18370051
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Functional biochemistry
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| Research Institution | Tohoku University |
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Principal Investigator |
SOGAWA Kazuhiro Tohoku University, Tohoku University, Graduate School of Life Sciences, Professor (80175421)
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| Co-Investigator(Kenkyū-buntansha) |
FUKUMURA Hiroshi Tohoku University, Graduate school of Life Sciences, Professor (50208980)
YASUMOTO Ken-ichi Tohoku University, Graduate School of Life Sciences, Associate Professor (90241629)
TAKASAKI Chikahisa Tohoku University, Graduate School of Life Sciences, Assistant Professor (10004491)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥15,840,000 (Direct Cost: ¥14,400,000、Indirect Cost: ¥1,440,000)
Fiscal Year 2007: ¥6,240,000 (Direct Cost: ¥4,800,000、Indirect Cost: ¥1,440,000)
Fiscal Year 2006: ¥9,600,000 (Direct Cost: ¥9,600,000)
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| Keywords | FLIM-FRET / Elongin B / Elongin C / pVHL / Conformation Change / Protein-protein interaction / VBC複合体 / ストレス応答 |
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| Research Abstract |
The tumor suppressor gene VHL product forms a complex with elongin B and elongin C, and acts as a recognition subunit of a ubiquitin E3 ligase. Interactions between components in the complex were investigated in living cells by FRET-FLIM. Elongin B-Celurean or Celurean-elongin B was coexpressed with elongin C-Citrine or Citrine-elongin C in CHO-K1 cells. FRET signals were examined by measuring a change in the fluorescence lifetime of donors and by monitoring a corresponding fluorescence rise of acceptors. Clear FRET signals between elongin B and elongin C were observed in all combinations except for the combination of elongin B-Cerulean and Citrine-elongin C. Although similar experiments to examine interaction between pVHL30 and elongin C linked to Cerulean or Citrine were performed, FRET signals were rarely observed among all the combinations. However, the signal was greatly increased by coexpression of elongin B. These results together with results of coimmunoprecipitation experiment using pVHL, elongin C and elongin B suggest that a conformational change of elongin C and/or pVHL was induced by binding of elongin B. The conformational change of elongin C was investigated by measuring changes in the intramolecular FRET signal of elongin C liked to Cerulean and Citrine at its N- and C-terminus, respectively. A strong FRET signal was observed in the absence of elongin B, and this signal was modestly increased by coexpression of elongin B, demonstrating that a conformation change of elongin C was induced by the binding of elongin B.
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Report
(3 results)
Research Products
(14 results)
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[Journal Article] Identification of amino acid residues in the Ah receptor involved in ligand binding2007
Author(s)
K. Goryo, A. Suzuki, C. A. Del Carpio, K. Siizaki, E. Kuriyama, Y. Mikami, K. Kinoshita, K. Yasumoto, A. Ranpug, A. Miyamoto, Y. Fujii-Kuriyama, and K. Sogawa
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Journal Title
Biophys. Biochem. Res. Commun. 354
Pages: 396-402
Description
「研究成果報告書概要(欧文)」より
Related Report
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[Presentation] FLIM-FRET analysis of Arnt in the nucleus of living culture cells2007
Author(s)
K. Goryo, K. Kinoshita, F. Shimada, Y. Tomokuni, M. Takada, H. Fukumura, K. Sogawa
Organizer
The 30^<th> Annunal Meeting of the Molecular Biology Society of Japan. The 80^<th> Annual Meeting of the Japanese Biochemical Society
Place of Presentation
Pacifico Yokohama, InterContinental Yokohama Grand
Description
「研究成果報告書概要(欧文)」より
Related Report
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